Giardia duodenalis (syn. G. lamblia, G. intestinalis) is a pathogenic protozoan parasite, and in the United States and abroad, is a leading cause of intestinal disease in waterborne outbreaks, nursery or day-care facilities, and homosexuals. Fecal-oral transmission of the disease occurs when viable cysts are ingested, and excystation occurs in the upper small intestine with the emergence of two trophozoites. Trophozoites colonize the small intestine where, at least in part stimulated by bile, they form new cysts (encystment) which are shed in the feces. During encystment, Giardia trophozoites synthesize a filamentous cyst wall which is composed, in large part, of the amino sugar, N-acetylgalactosamine (GalNAc) which is undetectable in non-encysting trophozoites. In order to accomplish this, Giardia trophozoites use a synthetic pathway of inducible enzymes to convert endogenous glucose to GalNAc. This pathway is not only paramount in their cytodifferentiation from trophozoite to cyst, but represents a potential point of chemotherapeutic attack against Giardia. Several drugs are currently being used to treat giardiasis and many have unpleasant side effects. Some, including the most frequently used drug, metronidazole, are at risk of becoming obsolete. While resistance to metronidazole has not yet become a problem in practice, in vivo and in vitro resistance has been demonstrated and thus a potential problem exists. Since we have only recently discovered the inducible GalNAc synthetic pathway in Giardia, little is known of the regulation or genetic control of the enzymes that comprise it. Indeed, since discovery of this pathway, we have also found a previously undescribed enzyme activity, tentatively referred to as "cyst wall synthetase". "Cyst wall synthetase" activity appears to fix GalNAc into the insoluble filaments that compose the outer cyst wall of Giardia. It is our intent in this proposal to continue studying this pathway by 1) purifying, characterizing and localizing the inducible, particulate "cyst wall synthetase" and 2) determining the chemical nature of its uncharacterized product which is likely to be a major component of the Giardia cyst wall. We will employ enzyme and protein purification techniques to achieve the enzymatic aim and standard methods of carbohydrate analyses to achieve the chemical analysis of the enzyme's product. Since there is a paucity of information of the mechanisms of encystment in most protozoa, the detailed information generated here will provide important information about a key metabolic pathway vital to Giardia encystment and transmission (and perhaps related to that in other cyst~producing protozoa), and will likely provide us with information important to improving chemotherapy in the future.